Bottom Line:
A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP.Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized.Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

ABSTRACTOur knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

Figure 9: Spatial segregation of transcription foci and replication forks tracked by SeqA in fast-growing cells. (A) Images of SeqA, RNAP, DNA (nucleoid), overlays of SeqA (red) and DNA (green), and RNAP (green) and SeqA (red) from a representative fast-growing E. coli cell, as described in the legend to Figure 7. SeqA foci and transcription foci are largely located at different positions (red and green colors on the overlay of SeqA and RNAP). Note also that most of the SeqA foci appear to be separated from high intensities of DNA signals in the nucleoids (red and green colors on the overlay of SeqA and DNA). (B) The distribution of apparent SeqA-mCherry foci in a population of fast-growing cells. The red line in the histogram indicates the median number of SeqA foci in these cells. (C) Cumulative distribution of the distances between SeqA foci and their closest RNAP foci in the population of cells. (—-) SeqA-mCherry RNAP-Venus, and (- - -) SeqA-mCherry RNAP-Venus random. The gray rectangle represents the colocalization area (≤140 nm). Only 21.5% of the SeqA foci are within 140 nm of the closest transcription foci. Adapted from Cagliero et al. (2014).

Mentions:
Recent SIM co-images of RNAP-Venus with DNA as well as with SeqA-mCherry or SSB-mCherry serving as proxies for replisomes have revealed chromosome landscapes for the two major functions of transcription and replication, which could explain why they remain in harmony in fast-growing cells. The major transcription machinery and replisome are mostly located in different chromosome territories or spatially segregated in the nucleoid (Cagliero et al., 2014). For example, Figure 9A shows the SIM images of SeqA and its spatial relationship with DNA (DNA/SeqA overlay) and with RNAP (RNAP/SeqA) in a typical fast-growing cell (LB at 37°C). Similar to RNAP foci and foci of NusA/NusB, SeqA foci also are located at the periphery of the nucleoid (DNA/SeqA overlay), indicating that the replisome is also compartmentalized in regions low in density of DNA or at DNA loops. Image analyses of populations of fast-growing cells showed that on average, each cell contains 10 SeqA foci (Figure 9B). In contrast to NusA/NusB, the cumulative distribution of SeqA foci and RNAP foci in the population of fast-growing cells has shown that most (~80%) of the SeqA foci are not colocalized with the RNAP foci, i.e., the two cellular functions are mostly segregated in space (Figure 9C). The low co-localization frequency of SeqA foci and RNAP foci suggests transient overlapping of transcription and replication of rrn regions. It is conceivable that during replication of rrn operons, RNAP foci are somehow disassembled, allowing replication forks to pass through the region, followed by reassembly of transcription machinery at the rrn clusters. Development of fast, super-resolution, time-lapse, live-cell imaging techniques will be necessary to address the dynamic interaction and segregation of the two active cellular functions in fast-growing cells.

Figure 9: Spatial segregation of transcription foci and replication forks tracked by SeqA in fast-growing cells. (A) Images of SeqA, RNAP, DNA (nucleoid), overlays of SeqA (red) and DNA (green), and RNAP (green) and SeqA (red) from a representative fast-growing E. coli cell, as described in the legend to Figure 7. SeqA foci and transcription foci are largely located at different positions (red and green colors on the overlay of SeqA and RNAP). Note also that most of the SeqA foci appear to be separated from high intensities of DNA signals in the nucleoids (red and green colors on the overlay of SeqA and DNA). (B) The distribution of apparent SeqA-mCherry foci in a population of fast-growing cells. The red line in the histogram indicates the median number of SeqA foci in these cells. (C) Cumulative distribution of the distances between SeqA foci and their closest RNAP foci in the population of cells. (—-) SeqA-mCherry RNAP-Venus, and (- - -) SeqA-mCherry RNAP-Venus random. The gray rectangle represents the colocalization area (≤140 nm). Only 21.5% of the SeqA foci are within 140 nm of the closest transcription foci. Adapted from Cagliero et al. (2014).

Mentions:
Recent SIM co-images of RNAP-Venus with DNA as well as with SeqA-mCherry or SSB-mCherry serving as proxies for replisomes have revealed chromosome landscapes for the two major functions of transcription and replication, which could explain why they remain in harmony in fast-growing cells. The major transcription machinery and replisome are mostly located in different chromosome territories or spatially segregated in the nucleoid (Cagliero et al., 2014). For example, Figure 9A shows the SIM images of SeqA and its spatial relationship with DNA (DNA/SeqA overlay) and with RNAP (RNAP/SeqA) in a typical fast-growing cell (LB at 37°C). Similar to RNAP foci and foci of NusA/NusB, SeqA foci also are located at the periphery of the nucleoid (DNA/SeqA overlay), indicating that the replisome is also compartmentalized in regions low in density of DNA or at DNA loops. Image analyses of populations of fast-growing cells showed that on average, each cell contains 10 SeqA foci (Figure 9B). In contrast to NusA/NusB, the cumulative distribution of SeqA foci and RNAP foci in the population of fast-growing cells has shown that most (~80%) of the SeqA foci are not colocalized with the RNAP foci, i.e., the two cellular functions are mostly segregated in space (Figure 9C). The low co-localization frequency of SeqA foci and RNAP foci suggests transient overlapping of transcription and replication of rrn regions. It is conceivable that during replication of rrn operons, RNAP foci are somehow disassembled, allowing replication forks to pass through the region, followed by reassembly of transcription machinery at the rrn clusters. Development of fast, super-resolution, time-lapse, live-cell imaging techniques will be necessary to address the dynamic interaction and segregation of the two active cellular functions in fast-growing cells.

Bottom Line:
A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP.Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized.Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

ABSTRACTOur knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.